Functional discontinuities in prothymosin ? caused by caspase cleavage in apoptotic cells

Abstract

Our study examines the effect of apoptosis on prothymosin alpha, an abundant, nuclear protein intimately involved with proliferation of all mammalian cells. When HeLa cells were treated with actinomycin D, with etoposide, or with staurosporine following synchronization with hydroxyurea, they underwent apoptosis based on several specific criteria, including fragmentation of DNA and activation of specific caspases. Similarly treated NIH3T3 cells arrested and displayed no indicators of apoptosis. In HeLa, but not in NIH3T3 cells, prothymosin alpha levels declined precipitously and a truncated version of the protein was formed. The following observations implicate caspase activity: (1) The truncated polypeptide arose only in the treated HeLa cell cultures. (2) The appearance of the truncated polypeptide coincided with the activation of caspase 3 and the cleavage of poly(ADP-ribose) polymerase, a known caspase substrate. (3) Carbobenzoxy-DEVD-fluoromethylketone, a cell-permeable caspase 3 inhibitor, blocked cleavage and degradation of prothymosin alpha. (4) The same inhibitor, when added to mixed extracts of apoptotic and normal cells, prevented cleavage of intact prothymosin alpha. (5) Recombinant caspase 3 and, to a much lesser extent, caspase 7 truncated purified prothymosin alpha. (6) In HeLa cells, cleavage occurred at three overlapping caspase 3-like sites with the consensus sequence D-X-X-D and released 10 to 14 residues from the carboxyl terminus, including the core nuclear localization signal. Two immediate consequences of the cleavage were observed: truncated prothymosin alpha was no longer confined to the nucleus and it was deficient in phosphate. These data suggest that the disabling of prothymosin alpha is a significant event in apoptosis. J. Cell. Physiol. 182:256-268, 2000. Published 2000 Wiley-Liss, Inc.

Stress-induced non-vesicular release of prothymosin-α initiated by an interaction with S100A13, and its blockade by caspase-3 cleavage

“…When rat ProTa was treated with active caspase-3, a 13 kDa protein band was time dependently generated in a zDEVD-fmk-reversible manner (Figure 6c). This finding is consistent with reports that human ProTa has three overlapping caspase-3 cleavage sites, 94DDED97, 95DEDD98, and 97DDVD101, 20,21 immediately upstream of the NLS moiety in its C-terminus, and that this fragment lacks an NLS moiety KKQK, which is present in rat ProTa at amino-acid positions 103-106. To identify the cleavage sites of rat ProTa by active caspase-3, we performed MALDI-TOF analysis.…”

“…4 Furthermore, there is a report that ProTa is released from the nuclei when the NLS is cleaved off by caspase-3. 20,21 This study clearly showed that the C-terminal region of ProTa including NLS is cleaved in culture by the apoptosis-induced compound, and in cell-free digestion of recombinant ProTa by active caspase-3 (Figure 6d-g). As ProTa devoid of C-terminal region (a.a.98 or 99-112) is conceived to lose the activity of interaction with S100A13 (Figure 4d), it will remain in the cytosol without extracellular release.…”

Prothymosin α Is Processed to Thymosin α1 and Thymosin α11 by a Lysosomal Asparaginyl Endopeptidase

“…Proteolysis of ProT␣ at the Asp 99 residue by caspase 3 has recently been reported to occur in HeLa cells in which apoptosis has been induced (35,36). This proteolysis has so far not been demonstrated to be a generalized process occurring in other cell types undergoing apoptosis and, in any case, occurs in an entirely different biological context (i.e.…”

Prothymosin Alpha and Immune Responses

“…In contradiction, many studies have also supported proTα's cytoplasmic localization (Sburlati, Manrow, & Berger, 1990;Tsitsiloni, Yialouris, Sekeri-Pataryas, & Haritos, 1989). In an effort to address this antiphasis, Enkemann and coworkers demonstrated that proTα is in principle detected at active transcription sites in the nucleus, while a smaller fraction remains in the cytoplasm (Enkemann, Wang, Trumbore, & Berger, 2000). It was additionally suggested that, owing to its small size and its negative charge, proTα may facilitate the movement of other positively charged molecules (eg, of histones) into and within the nucleus, particularly in highly charged environments where there is a need to overcome electrostatic interactions.…”